This invention relates generally to methods and apparatus for conducting amplification and various analyses of polynucleotides. More particularly, the invention relates to the design and construction of small, typically single-use, modules for use in analyses involving polynucleotide amplification reactions such as the polymerase chain reaction (PCR).
In recent decades, the art has developed a very large number of protocols, test kits, and cartridges for conducting analyses of biological samples for various diagnostic and monitoring purposes. Immunoassays, immunometric assays, agglutination assays and analyses based on polynucleotide amplification assays (such as polymerase chain reaction), or on various ligand-receptor interactions and/or differential migration of species in a complex sample, all have been used to determine then presence or concentration of various biological compounds or contaminants, or the presence of particular cell types.
Recently, small, disposable devices have been developed for handling biological samples and for conducting certain clinical tests. Shoji et al. reported the use of a miniature blood gas analyzer fabricated on a silicon wafer. Shoji et al., Sensors and Actuators, 15:101-107 (1988). Sato et al. reported a cell fusion technique using micromechanical silicon devices. Sato et al., Sensors and Actuators, A21-A23:948-953 (1990). Ciba Corning Diagnostics Corp. (USA) has manufactured a microprocessor-controlled laser photometer for detecting blood clotting.
Micromachining technology, using, e.g., silicon substrates, has enabled the manufacture of microengineered devices having structural elements with minimal dimensions ranging from tens of microns (the dimensions of biological cells) to nanometers (the dimensions of some biological macromolecules). Angell et al., Scientific American, 248: 44-55 (1983). Wise et al., Science, 254:1335-42 (1991); and Kricka et al., J. Int. Fed. Clin. Chem., 6:54-59 (1994). Most experiments involving structures of this size relate to micromechanics, i.e., mechanical motion and flow properties. The potential capability of these structures has not been exploited fully in the life sciences.
Brunette (Exper. Cell Res., 167:203-217 (1986) and 164:11-26 (1986)) studied the behavior of fibroblasts and epithelial cells in grooves in silicon, titanium-coated polymers and the like. McCartney et al. (Cancer Res., 41:3046-3051 (1981)) examined the behavior of tumor cells in grooved plastic substrates. LaCelle (Blood Cells, 12:179-189 (1986)) studied leukocyte and erythrocyte flow in microcapillaries to gain insight into microcirculation. Hung and Weissman reported a study of fluid dynamics in micromachined channels, but did not produce data associated with an analytic device. Hung et al., Med. and Biol. Engineering, 9:237-245 (1971); and Weissman et al., Am. Inst. Chem. Eng. J., 17:25-30 (1971). Columbus et al. utilized a sandwich composed of two orthogonally orientated v-grooved embossed sheets in the control of capillary flow of biological fluids to discrete ion-selective electrodes in an experimental multi-channel test device. Columbus et al., Clin. Chem., 33:1531-1537 (1987). Masuda et al. and Washizu et al. have reported the use of a fluid flow chamber for the manipulation of cells (e.g., cell fusion). Masuda et al., Proceedings IEEE/IAS Meeting, pp. 1549-1553 (1987); and Washizu et al., Proceedings IEEE/IAS Meeting pp. 1735-1740 (1988). Silicon substrates have been used to develop microdevices for pH measurement and biosensors. McConnell et al., Science, 257:1906-12 (1992); and Erickson et al., Clin. Chem., 39:283-7 (1993). However, the potential of using such devices for the analysis of biological fluids heretofore has remained largely unexplored.
Methodologies for using polymerase chain reaction (PCR) to amplify a segment of DNA are well established. (See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, pp. 14.1-14.35.) A PCR amplification reaction can be performed on a DNA template using a thermostable DNA polymerase, e.g., Taq DNA polymerase (Chien et al. J. Bacteriol., 127:1550 (1976)), nucleoside triphosphates, and two oligonucleotides with different sequences, complementary to sequences that lie on opposite strands of the template DNA and which flank the segment of DNA that is to be amplified (xe2x80x9cprimersxe2x80x9d). The reaction components are cycled between a higher temperature (e.g., 94xc2x0 C.) for dehybridizing (xe2x80x9cmeltingxe2x80x9d) double stranded template DNA, followed by lower temperatures (e.g., 40-60xc2x0 C. for annealing of primers and, e.g., 70-75xc2x0 C. for polymerization). A repeated reaction cycle between dehybridization, annealing and polymerization temperatures provides approximately exponential amplification of the template DNA. For example, up to 1 xcexcg of target DNA up to 2 kb in length can be obtained from 30-35 cycles of amplification with only 10xe2x88x926 xcexcg of starting DNA. Machines for performing automated PCR chain reactions using a thermal cycler are available (Perkin Elmer Corp.)
Polynucleotide amplification has been applied to the diagnosis of genetic disorders (Engelke et al., Proc. Natl. Acad. Sci., 85:544 (1988), the detection of nucleic acid sequences of pathogenic organisms in clinical samples (Ou et al., Science, 239:295 (1988)), the genetic identification of forensic samples, e.g., sperm (Li et al., Nature, 335:414 (1988)), the analysis of mutations in activated oncogenes (Farr et al., Proc. Natl. Acad. Sci., 85:1629 (1988)) and in many aspects of molecular cloning (Oste, Biotechniques, 6:162 (1988)). Polynucleotide amplification assays can be used in a wide range of applications such as the generation of specific sequences of cloned double-stranded DNA for use as probes, the generation of probes specific for uncloned genes by selective amplification of particular segments of cDNA, the generation of libraries of cDNA from small amounts of mRNA, the generation of large amounts of DNA for sequencing, and the analysis of mutations.
A wide variety of devices and systems has been described in the art for conducting polynucleotide amplification reactions using thermal cycling procedures. Templeton, Diag. Mol. Path., 1:58-72 (1993); Lizardi et. al., Biotechnology, 6:1197-1202 (1988); Backman et al., Eur. Patent No. 320308 (1989); and Panaccio et al., BioTechniques, 14:238-43 (1993). The devices use a wide variety of design principles for transfer, such as water baths, air baths and dry blocks such as aluminum. Haff et al., BioTechniques, 10:102-12 (1991); Findlay et al., Clin. Chem., 39:1927-33 (1993); Wittwer et al., Nucl. Acids Res., 17:4353-7 (1989). PCR reactions in small reaction volumes have been described. Wittwer et al., Anal. Biochem., 186:328-31 (1990); and Wittwer et al., Clin. Chem., 39:804-9 (1993). Polynucleotide amplification micro-devices fabricated from silicon also have been described. Northrup et al., in: Digest of Technical Papers: Transducers 1993 (Proc. 7th International Conference on Solid State Sensors and Actuators) Institute of Electrical and Electronic Engineers, New York, N.Y., pp. 924-6; and Northrup et al., PCT WO 94/05414 (1994).
Silica particles have been shown to bind to nucleic acids, and have been used to isolate nucleic acids prior to PCR analysis. Zeillinger et al., BioTechnigues, 14:202-3 (1993). While the art has described the use of silicon and other substrates fabricated with microchannels and chambers for use in a variety of analyses, little attention has been focused on methods for the modification of micromachined silicon or other surfaces, to diminish binding or other properties of the surfaces, which can inhibit reactions, such as polynucleotide amplification reactions, conducted in the devices. Northrup et al. describe the chemical silanization of a PCR reaction chamber in a silicon substrate having a depth of 0.5 mm. Northrup et al., in: Digest of Technical Papers: Transducers 1993 (Proc. 7th International Conference on Solid State Sensors and Actuators) Institute of Electricar and Electronic Engineers, New York, N.Y., pp. 924-6; and Northrup et al., PCT WO 94/05414 (1994). The reference of Northrup et al., (in: Digest of Technical Papers:Transducers 1993), however, discloses that, in the absence of silanization, untreated silicon surfaces of the reaction chambers had no inhibitory effect on the PCR reaction.
There is a need for convenient, rapid systems for polynucleotide amplification analyses, which could be used clinically in a wide range of potential applications in clinical tests such as tests for paternity, and genetic and infectious diseases and a wide variety of other tests in the environmental and life sciences. There is a need for the development of micro-devices fabricated in substrates such as silicon which permit polynucleotide amplification reactions to be conducted in high yields without interfering effects on the reaction caused by the surfaces of the substrate.
An object of the invention is to provide microscale analytical devices with optimal reaction environments for conducting polynucleotide amplification reactions which can be used to detect very low concentrations of a polynucleotide and to produce analytical results rapidly. Another object is to provide easily mass produced, disposable, small (e.g., less than about 1 cc in volume) devices having functional elements capable of rapid, automated polynucleotide amplification analyses of a preselected cell or cell-free sample, in a range of applications. It is a further object of the invention to provide agents for use in microscale reaction chambers fabricated in solid substrates such as silicon, to diminish potential inhibitory effects of the substrate surfaces on a polynucleotide amplification reaction. It is a further object of the invention to provide apparatus for delivering reagents and sample fluids to and from microscale polynucleotide amplification chambers fabricated in solid substrates such as silicon, and to provide apparatus for sealing the reaction chamber during an amplification reaction. It is yet another object of the invention to provide apparatus that can be used to implement a range of rapid clinical tests, e.g., tests for viral or bacterial infection, tests for cell culture contaminants, or tests for the presence of a recombinant DNA or a gene in a cell, and the like.
These and other objects and features of the invention will be apparent from the description, drawings and claims which follow.
The invention provides a family of small, mass produced, typically one-use devices (sometimes referred to herein as xe2x80x9cchipsxe2x80x9d) for conducting a reaction to enable the rapid amplification of a polynucleotide in a sample. In one embodiment, the device comprises a solid substrate that is fabricated to comprise a mesoscale polynucleotide amplification reaction chamber. The device also may include a cover, e.g., a transparent cover, disposed over the substrate, to seal at least a portion of the reaction chamber during an amplification reaction. The device further includes at least one port in fluid communication with the reaction chamber, for introducing a sample into the chamber (sometimes referred to herein as a xe2x80x9csample inlet portxe2x80x9d or inlet portxe2x80x9d). The device may include one or more flow channels extending from the ports to the reaction chamber, and/or connecting two or more reaction chambers. The device also may include one or more additional ports in fluid communication with the reaction chamber, to serve as access ports, inlet/outlet ports and/or vents. One or more ports and/or flow channels of the device may be fabricated in the cover or in the substrate. In the device, the reaction chamber may be provided with a composition which diminishes inhibition of a polynucleotide amplification reaction by the wall(s) defining the reaction chamber. The device may also include means for thermally cycling the contents of the chamber to permit amplification of a sample polynucleotide.
The term xe2x80x9cmesoscalexe2x80x9d is used herein with reference to reaction chambers or flow channels, at least one of which has at least one cross-sectional dimension between about 0.1 xcexcm and 1,000 xcexcm. The flow channels leading to the reaction chamber have preferred widths and depths on the order of about 2.0 to 500 xcexcm. Chambers in the substrate wherein amplification takes place may have one or more larger dimensions, e.g., widths and/or lengths of about 1 to 20 mm. Preferred reaction chamber widths and lengths are on the order of about. 5 to 15 mm. The reaction chambers are fabricated with depths on the order of about 0.1 to at most about 1,000 xcexcm. Typically, the reaction chambers are fabricated with depths less than 500 xcexcm, e.g., less than about 300 xcexcm, and optionally less than about 80 xcexcm. Fabrication of the reaction chamber, with shallow depths, e.g., less than 300 xcexcm, advantageously facilitates heat transfer to the reaction chamber contents, e.g., through the substrate, and permits efficient thermal cycling during an amplification reaction requiring thermal cycling. However, in some embodiments, the reaction chambers may be fabricated with depths between about 500 xcexcm and 1,000 xcexcm. The overall size of the device ranges from microns to a few millimeters in thickness, depending on the material from which it is constructed, and approximately 0.2 to 5.0 centimeters in length or width.
The devices may be used to amplify and/or analyze microvolumes of a sample, introduced into the flow system through an inlet port defined, e.g., by a hole communicating through the substrate or the cover. The volume of the mesoscale flow system typically will be less than 50 xcexcl, and the volume of the reaction chambers is often less than 20 xcexcl, e.g., 10 xcexcl or less. The volume of the individual channels and chambers in another embodiment may be less than 1 xcexcl, e.g., in the nanoliter or picoliter range. Polynucleotides present in very low concentrations, (e.g., nanogram quantities) can be rapidly amplified (e.g., in less than ten minutes) and detected. After a polynucleotide amplification assay is complete, the devices may be discarded or they may be cleaned and re-used.
In one embodiment, reaction chambers may be fabricated wherein the ratio of the surface area of the walls defining the reaction chamber to the volume of the reaction chamber is greater than about 3 mm2/xcexcl. Chambers also may be fabricated with even higher surface area to volume ratios, such as 5 mm2/xcexcl or, optionally, greater than 10 mm2/xcexcl. As the ratio of the surface area to volume increases, heat transfer through the substrate to and from the reaction chamber contents is facilitated, and thus thermal cycling of the reaction becomes more efficient, and the productivity of the reaction is increased. Additionally, however, as the ratio of the surface area to volume increases, potential inhibitory effects of the walls of the substrate on the polynucleotide amplification reaction are increased. Depending on the material from which the device is made, the wall surfaces of the mesoscale channels and chambers could interfere with the polynucleotide amplification, e.g., via binding interactions between the material and sample polynucleotides or amplification reagents.
The invention provides a range of compositions which may be provided in the reaction chamber to diminish the potentially inhibitory effects of the reaction chamber wall surfaces, such as silicon surfaces, on the reaction. The compositions are particularly useful in reaction chambers having a surface area to volume ratio greater than about 3 mm2/xcexcl or 5 mm2/xcexcl, or, in another embodiment, in chambers wherein the ratio exceeds about 10 mm2/xcexcl. The device also may include a cover disposed over the reaction chamber to seal the reaction chamber during an amplification reaction. The cover may comprise a material such as glass or silicon, or a plastic material. The use of a cover disposed over the reaction chamber increases the total amount of surface area in contact with fluid in the reaction chamber. The surface of the cover exposed to the reaction chamber also may be treated with compositions as disclosed herein to reduce potentially inhibitory effects of the cover surface material on the amplication reaction.
A composition provided in the reaction chamber to diminish inhibition of an amplification reaction by a wall of the reaction chamber may be covalently or non-covalently adhered to the surface of the reaction chamber wall, or may be provided in solution in the reaction chamber during an amplification reaction. In one embodiment, the wall surfaces of one or more reaction chamber(s) and/or channel(s) in the device may be coated with a silane, using a silanization reagent such as dimethychlorosilane, dimethydichlorosilane, hexamethyldisilazane or trimethylchlorosilane (available, e.g., from Pierce, Rockford, Ill.). Alternatively, the surface of the walls of the reaction chamber(s) and/or the flow channel(s), e.g., fabricated within a silicon substrate, may be provided with a relatively inert coating, for example, using a siliconizing reagent, such as Aquasil(trademark) or Surfasil(trademark) (Pierce, Rockford, Ill.), or Sigmacote(trademark) (Sigma Chemical Co., St. Louis, Mo.). Siliconizing reagents available from commercial manufacturers, such as Pierce (Rockford, Ill.) or Sigma Chemical Co. (St. Louis, Mo.), are organosilanes containing a hydrolyzable group, which can hydrolyze in solution to form a silanol which can polymerize and form a film over the surface of the chamber, and can react with hydroxyl groups on the surface of the chamber, such that the film is tightly bonded over the entire surface. The coating may further include a macromolecule (sometimes referred to herein as a xe2x80x9cblocking agentxe2x80x9d) noncovalently or covalently associated with the silicone coating, to further reduce inhibitory effects of the wall of the reaction chamber on the amplification reaction. Useful macromolecules include an amino acid polymer, or polymers such as polyvinylpyrrolidone, polyadenylic acid and polymaleimide.
A silicon oxide film may be provided on the surface of the reaction chamber and/or channel walls, in a silicon substrate, to reduce inhibition of the amplification reaction by the wall surfaces. The silicon oxide film may be formed by a thermal process wherein the silicon substrate is heated in the presence of oxygen. Alternatively, a plasma-enhanced oxidation or plasma-enhanced chemical vapor deposition process may be utilized. Additionally the reaction chamber and/or channel walls may be coated with a relatively inert polymer such as a poly (vinyl chloride).
Prior to addition of the sample polynucleotide and amplification reagents to the reaction chamber, another polynucleotide (sometimes referred to herein as a xe2x80x9cblockingxe2x80x9d polynucleotide) may be added to the chamber, such as genomic DNA or polyadenylic acid, preferably at a concentration greater than the concentration of the sample polynucleotide. This permits the blocking polynucleotide to occupy any sites on the wall surfaces that could potentially bind to the sample polynucleotide and reduce the yield of the reaction or the precision of the assay. Thus, in one embodiment, a blocking polynucleotide may be provided in a reaction chamber fabricated within a silicon substrate, such that the blocking polynucleotide may occupy any polynucleotide binding sites, such as free hydroxyl groups, on the wall surfaces of the reaction chamber. To avoid interfering with the amplification reaction, the blocking polynucleotide should comprise sequences unrelated to that of the sample polynucleotide. Other compositions which bind to the chamber wall surfaces, such as polyguanylic acid or various polypeptides such as casein or serum albumin, could also be utilized as a blocking agent.
The devices may be utilized to implement a polynucleotide amplification reaction, such as a polymerase chain reaction (PCR), in the reaction chamber. The reaction chamber may be provided with reagents for PCR including a sample polynucleotide,polymerase, nucleoside triphosphates, a first primer hybridizable with the sample polynucleotide, and a second primer hybridizable with a sequence that is complementary to the sample polynucleotide, wherein the first and second primers define the termini of the amplified polynucleotide product. The device also may include means for thermally cycling the contents of the amplification reaction chamber, such that, in each cycle, e.g., the temperature is controlled to 1) dehybridize (xe2x80x9cmeltxe2x80x9d) double stranded polynucleotide, 2) anneal the primers to single stranded polynucleotide, and 3) synthesize amplified polynucleotide between the primers. Other amplification methods available in the art also may be utilized, including, but not limited to: (1) target polynucleotide amplification methods such as self-sustained sequence replication (3SR) and strand-displacement amplification (SDA); (2) methods based on amplification of a signal attached to the target DNA, such as xe2x80x9cbranched chainxe2x80x9d DNA amplification (Chiron Corp.); (3) methods based on amplification of probe DNA, such as ligase chain reaction (LCR) and QB replicase amplification (QBR); and (4) various other methods such as ligation activated transcription (LAT), nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR) and cycling probe reaction (CPR) (for a review of these methods, see pp. 2-7 of The Genesis Report, DX, Vol. 3, No. 4, Feb. 1994; Genesis Group, Montclair, N.J.).
The reaction chamber may be fabricated with one section which is thermally cycled sequentially between the required temperatures for polynucleotide amplification reactions requiring thermal cycling, such as conventional PCR. Alternatively, the reaction chamber may comprise two or more sections, set at the different temperatures required for dehybridization, annealing and polymerization, in which case the device further comprises means for transferring the contents of the chamber between the sections to implement the reaction, e.g., a pump controlled by a computer. The reaction chamber may be bound in at least a portion of the chamber by a cover disposed over the substrate. The device may further include means for detecting the amplified polynucleotide, as disclosed herein. The devices may be used to implement a variety of automated, sensitive and rapid polynucleotide analyses, including analyses for the presence of polynucleotides in cells or in solution, or for analyses for a virus or cell types using the presence of a particular polynucleotide as a marker.
The mesoscale flow channel(s) and reaction chamber(s) may be designed and fabricated from solid substrates using established micromachining methods such as photolithography, etching and disposition techniques, laser machining, LIGA processing (Becker et al., Microelec. Eng. 4: 35-56, 1986) and plastic molding. The mesoscale flow systems in the devices may be constructed by fabricating flow channels and one or more reaction chambers into the surface of the substrate, and then adhering or clamping a cover over the surface. The solid substrate and/or cover may comprise a material such as silicon, polysilicon, silica, glass, gallium arsenide, polyimide, silicon nitride and silicon dioxide. The cover and/or the substrate alternatively may comprise a plastic material such as an acrylic, polycarbonate polystyrene or polyethylene. Optionally the cover and/or substrate may comprise a transparent material.
An appliance also may be provided, for use with the device, which contains a nesting site for holding the substrate of the device and which optionally mates one or more input ports on the substrate with one or more. flow lines in the appliance. After a biological fluid sample suspected to contain a particular polynucleotide is applied to the inlet port, the substrate is placed in the appliance and pumps, e.g., disposed in the appliance, are actuated to force the sample through the flow system. Alternatively, a sample may be injected into the substrate by the appliance (e.g. by a syringe fitted to the appliance). Reagents required for the assay, such as a polymerase enzyme, may be added (in liquid or in dry form) to the polynucleotide sample prior to injection into the substrate. Alternatively, reagents necessary to complete the assay can be injected into the reaction chamber from a separate inlet port, e.g., by the appliance. Fluid samples and reagents may also enter the mesoscale flow system by capillary action or by gravity.
The invention also provides means for sealing one or more of the fluid inlet/outlet ports in the device during an amplification reaction. This advantageously prevents evaporation of liquids during thermal cycling and thus maintains the preferred reaction concentrations during the amplification reaction. In one embodiment, an apparatus including means for delivering fluid to and from the reaction chamber through a port in the device, and adapted to interfit and/or interlock with the port is provided, which can reversibly seal the port after delivery of fluid to the reaction chamber. For example, the fluid delivery apparatus may comprise a syringe or pipette. In one embodiment, the fluid delivery apparatus may comprise a pipette including a pipette tip provided with an aperture for transferring fluid between the pipette and the port. The pipette tip optionally may be releasable from the pipette, and may be disposable to prevent contamination between samples.
The device may include a substrate comprising a heat conducting material such as silicon, as well as a cover disposed over the substrate, which may comprise a transparent material such as glass or a plastic. The device also includes the mesoscale polynucleotide amplification chamber, fabricated within the substrate or the cover. The cover may include a cavity for receiving and interfitting with the pipette used to deliver sample and reagent solutions to and from the reaction chamber. The device may further include a flow channel that communicates through the substrate and/or the cover between the aperture of the pipette tip and the reaction chamber, when the pipette is fitted within the cavity. The aperture may be positioned. on a wall of the pipette tip to permit the pipette tip to move between a first position which permits transfer of fluid from the tip through the aperture and the channel to the reaction chamber, and a second position to permit the aperture to face a wall of the cavity, thereby to seal the flow channel and the reaction chamber during a reaction. Additionally, a depressible member may be provided which extends from the substrate and can seal the port upon depression of the member against the port.
The temperature of one or more section(s) in the reaction chamber can be regulated by, e.g., providing one or more electrical resistance heaters in the substrate near the reaction chamber, or by using a pulsed laser or other source of electromagnetic energy directed to the reaction chamber. The appliance may include electrical contacts in the nesting region which mate with contacts integrated into the structure of the substrate, e.g., to power electrical resistance heating of the reaction chamber. A cooling element may also be provided in the appliance, to assist in the thermal regulation of the reaction chamber. The appliance may be provided with conventional circuitry in communication with sensors in the device for thermally regulating the temperature cycles required for the dehybridization and polymerization reactions.
The amplified polynucleotide produced by the polynucleotide amplification reaction in the mesoscale reaction chamber can be collected through a port in the substrate and detected. Alternatively, specific reagents and methods known in the art may be employed to directly detect amplification products in the reaction chamber (xe2x80x9cTaq Man(trademark)xe2x80x9d PCR reagents and kit, available from Perkin Elmer Corp., for example). As another alternative, a mesoscale detection region may be microfabricated in the substrate, in fluid communication with the reaction chamber in the device, as a part of the mesoscale flow system. The detection region may include a labeled binding moiety, such as a labeled polynucleotide or antibody probe, capable of detectably binding with the amplified polynucleotide. The presence of polymerized polynucleotide product in the detection region can be detected, e.g., by optical detection of agglutination of the polymerized polynucleotide and the binding moiety through a glass cover over the detection region or through a translucent or transparent section of the substrate itself. Alternatively, the detection region may comprise a series of channels or microlithographic arrays for electrophoretically separating and detecting an amplified polynucleotide.
A positive assay may also be indicated by detectable changes in sample fluid flow properties, such as changes in pressure or electrical conductivity at different points in the flow system upon production of amplified polynucleotide in the reaction chamber. In one embodiment, the device comprises a mesoscale flow system which includes a polynucleotide amplification reaction chamber, and a detection region (e.g., a chamber or a portion of a flow channel), used in combination with an appliance which includes sensing equipment such as a spectrophotometer capable of reading a positive result through an optical window, e.g., disposed over the detection region. The appliance may also be designed to receive electrical signals indicative of a pressure reading, conductivity, or the like, sensed in the reaction chamber, the detection region, or some other region of the flow system.
The substrate may comprise a plurality of reaction and/or detection chambers to enable the rapid parallel amplification and/or detection of several polynucleotides in a mixture. The mesoscale flow system may include protrusions, or a section of reduced cross-sectional area, to cause lysis of cells in the microsample prior to delivery to the reaction chamber. Sharp edged pieces of silicon, trapped in the flow path, can be used as a lysis means. The mesoscale flow system also may include a cell capture region comprising a binding moiety, e.g., immobilized on a wall of a flow channel, which binds a particular type of cell in a heterogeneous cell population at a relatively low fluid flow rate, and at a greater flow rate or by changing the nature of the solvent, for example, releases the cell type prior to delivery of the cells to a cell lysis region, then to a reaction chamber. In this embodiment, intracellular DNA or RNA is isolated from a selected cell subpopulation and delivered to the mesoscale reaction chamber for polynucleotide analysis in one device. In an alternative embodiment, the binding reagent may by immobilized on a solid particle, such as a latex or magnetic bead, as described below.
Complex-forming agents, such as magnetic beads coated with a polynucleotide probe, may be provided within the mesoscale flow system, which can be moved along the flow system by an external magnetic field, e.g., in the appliance. The polynucleotide probe immobilized on the magnetic beads enables the beads to bind to amplified polynucleotide in the reaction chamber or in a separate detection chamber. Magnetic beads containing an immobilized polynucleotide probe may be, e.g., carried through the flow system or otherwise introduced to the reaction chamber at the end of an assay to bind to the amplified polynucleotide product. The bound polynucleotide may then be transported on the magnetic beads to a detection or purification chamber in the flow system, or to a collection port. Alternatively, the magnetic beads may be held in place at a predetermined location in the device, then transported to a detection or purification chamber after binding the polynucleotide product.
Some of the features and benefits of the devices are illustrated in Table 1. The devices can provide a rapid test for the detection of pathogenic bacteria or viruses, or for the presence of certain cell types, or the presence of a gene or a recombinant DNA sequence in a cell. The devices as disclosed herein are all characterized by a mesoscale flow system including a polynucleotide amplification reaction chamber, preferably having at least one mesocale dimension, which is used to amplify a polynucleotide in a sample, and which may be provided with the required amplification reagents. The device may be used to amplify a polynucleotide in a wide range of applications. At the conclusion of the assay the device may be discarded, or it may be cleaned and re-used.